Mutation of RPE65 can cause severe blindness from birth or early childhood, and RPE65 protein is associated with retinal pigment epithelium (RPE) vitamin A metabolism. Here, we show that Rpe65-deficient mice exhibit changes in retinal physiology and biochemistry. Outer segment discs of rod photoreceptors in Rpe65-/- mice are disorganized compared with those of Rpe65+/+ and Rpe65+/- mice. Rod function, as measured by electroretinography, is abolished in Rpe65-/- mice, although cone function remains. Rpe65-/- mice lack rhodopsin, but not opsin apoprotein. Furthermore, all-trans-retinyl esters over-accumulate in the RPE of Rpe65-/- mice, whereas 11-cis-retinyl esters are absent. Disruption of the RPE-based metabolism of all-trans-retinyl esters to 11-cis-retinal thus appears to underlie the Rpe65-/- phenotype, although cone pigment regeneration may be dependent on a separate pathway.
Regeneration of 11-cis retinal from all-trans retinol in the retinal pigment epithelium (RPE) is a critical step in the visual cycle. The enzyme(s) involved in this isomerization process has not been identified and both all-trans retinol and all-trans retinyl esters have been proposed as the substrate. This study is to determine the substrate of the isomerase enzyme or enzymatic complex. Incubation of bovine RPE microsomes with all-trans [(3)H]-retinol generated both retinyl esters and 11-cis retinol. Inhibition of lecithin retinol acyltransferase (LRAT) with 10-N-acetamidodecyl chloromethyl ketone (AcDCMK) or cellular retinol-binding protein I (CRBP) diminished the generation of both retinyl esters and 11-cis retinol from all-trans retinol. The 11-cis retinol production correlated with the retinyl ester levels, but not with the all-trans retinol levels in the reaction mixture. When retinyl esters were allowed to form prior to the addition of the LRAT inhibitors, a significant amount of isomerization product was generated. Incubation of all-trans [(3)H]-retinyl palmitate with RPE microsomes generated 11-cis retinol without any detectable production of all-trans retinol. The RPE65 knockout (Rpe65(-/-)) mouse eyecup lacks the isomerase activity, but LRAT activity remains the same as that in the wild-type (WT) mice. Retinyl esters in WT mice plateau at 8 weeks-of-age, but Rpe65(-/-) mice continue to accumulate retinyl esters with age (e.g., at 36 weeks, the levels are 20x that of WT). Our data indicate that the retinyl esters are the substrate of the isomerization reaction.
To determine whether the photoreceptors change structurally and functionally during aging, and to analyze whether pigmentation in the retinal pigment epithelium might be a contributing factor. Young, adult, and aged C57BL/6 and Balb/c mice (1, 4, and 17 months of age) were housed under a 12-h light/12-h dark cycle, with an ambient light intensity at the eye level of the mice of 85 +/- 18 lux. Scotopic single-flash and photopic-flicker electroretinograms (ERGs) after complete dark adaptation were used to assess rod and cone function, respectively. Numbers of rod photoreceptors were counted in plastic sections, and rhodopsin levels were measured using absorption difference spectrophotometry. Numbers and types of cones were determined using lectin staining in retinal flatmounts and cone-specific antibodies in radial frozen sections. Young pigmented C57BL/6 and nonpigmented Balb/c mice had similar numbers of rods. In both mouse strains, there was an overall decline in rod photoreceptor number during aging, which was more pronounced in albino mice. Rod cell numbers correlated with a drop in the overall amount of rhodopsin and a reduction in the maximum a-wave of the rod ERG. The number of short-wavelength cones was unaffected by age and pigmentation, whereas an age-related decline was observed in mid-wavelength (MWL) cones in albino, but not in pigmented mice. In contrast, MWL cone function was reduced during aging in both strains. Flicker-fusion frequency was determined to be approximately 10 Hz lower in albino animals, which is due to prolonged b-waves in these ERGs. Age-related changes were found in both photoreceptor systems, rods and cones, and in both pigmented and nonpigmented mice. However, rod photoreceptors appear to be more susceptible to both aging and the lack of pigmentation, when compared to cones. These results may help as we begin to understand certain age-related retinal diseases.
Retinal rod and cone pigments consist of an apoprotein, opsin, covalently linked to a chromophore, 11-cis retinal. Here we demonstrate that the formation of the covalent bond between opsin and 11-cis retinal is reversible in darkness in amphibian red cones, but essentially irreversible in red rods. This dissociation, apparently a general property of cone pigments, results in a surprisingly large amount of free opsin--about 10% of total opsin--in dark-adapted red cones. We attribute this significant level of free opsin to the low concentration of intracellular free 11-cis retinal, estimated to be only a tiny fraction (approximately 0.1 %) of the pigment content in red cones. With its constitutive transducin-stimulating activity, the free cone opsin produces an approximately 2-fold desensitization in red cones, equivalent to that produced by a steady light causing 500 photoisomerizations s-1. Cone pigment dissociation therefore contributes to the sensitivity difference between rods and cones.
Rpe65 ؊/؊ mice produce minimal amounts of 11-cis-retinal, the ligand necessary for the formation of photosensitive visual pigments. Therefore, the apoprotein opsin in these animals has not been exposed to its normal ligand. The Rpe65 ؊/؊ mice contain less than 0.1% of wild type levels of rhodopsin. Mass spectrometric analysis of opsin from Rpe65 ؊/؊ mice revealed unusually high levels of phosphorylation in dark-adapted mice but no other structural alterations. Single flash and flicker electroretinograms (ERGs) from 1-month-old animals showed trace rod function but no cone response. B-wave kinetics of the single-flash ERG are comparable with those of dark-adapted wild type mice containing a full compliment of rhodopsin. Application (intraperitoneal injection) of 11-cis-retinal to Rpe65 ؊/؊ mice increased the rod ERG signal, increased levels of rhodopsin, and decreased opsin phosphorylation. Therefore, exogenous 11-cis-retinal improves photoreceptor function by regenerating rhodopsin and removes constitutive opsin phosphorylation. Our results indicate that opsin, which has not been exposed to 11-cis-retinal, does not generate the activity generally associated with the bleached apoprotein.RPE65 is a major protein in the retinal pigment epithelium (RPE) 1 (1) and has also been identified in cone photoreceptors (2, 3). The photoreceptors of Rpe65 Ϫ/Ϫ mice are almost completely depleted of 11-cis-retinal, the native ligand of cone and rod opsins, resulting in minimal levels of rhodopsin and the deterioration of their photosensitivity (4). Photoreceptor function has been shown to be partially restored by supplying exogenous ligand (5, 6). Thus, the Rpe65 Ϫ/Ϫ mouse is an excellent model in which to study the two key factors that control the activity of rhodopsin, the supply of the ligand (7, 8) and the level of rhodopsin/opsin phosphorylation. Therefore, we have analyzed opsin phosphorylation levels of Rpe65 Ϫ/Ϫ mice and correlated those levels with electroretinogram (ERG) responses and rhodopsin levels in the absence and presence of exogenous 11-cis-retinal.Retinal function is controlled by the availability of 11-cisretinal, which can be affected by the absence or dysfunction of any number of participants in the retinal metabolic pathway (e.g. Refs. 9 and 10). RPE65 is essential for production of 11-cis-retinoids (4). RPE65 mutations in humans result in congenital retinal dystrophies ranging from night blindness to loss of vision (11). Although night blindness would suggest loss of rod rather than cone function, the ERG retained in Rpe65 Ϫ/Ϫ mice appears to originate from rod photoreceptors (12).A second key factor that controls visual sensitivity is opsin/ rhodopsin phosphorylation. The C terminus of activated rhodopsin is multiply phosphorylated by rhodopsin kinase (G protein-coupled receptor kinase 1) (13) in vivo (14,15), and this multiple phosphorylation has been shown to be necessary for the rapid return of sensitivity (16). In moderate light levels, it has been proposed that opsin dephosphorylation and its regen...
In the aged Rpe65-/- mouse, opsin levels decrease because of the loss of photoreceptors. The remaining opsin is structurally intact, and the components of the phototransduction cascade and the retinal circuitry remain functional, despite the absence of normal photoreceptor activity.
As a method for sustained drug delivery, subcutaneous administration using Matrigel proved more efficacious than intraperitoneal injection for in vivo delivery of retinoids to cone photoreceptors. These experiments are the first to show a sustained delivery of retinoids in mice and suggest a strategy for potential clinical therapeutic development.
Rhodopsin is the dim light photosensitive pigment of animals. In this work, we undertook to study the structure of rhodopsin from swine and compare it with bovine and rat rhodopsin. Porcine rhodopsin was analyzed using methodology developed previously for mass spectrometric analysis of integral membrane proteins. Combining efficient protein cleavage and high performance liquid chromatography separation with the sensitivity of mass spectrometry (MS), this technique allows the observation of the full protein map and the posttranslational modifications of the protein in a single experiment. The rhodopsin protein from a single porcine eye was sequenced completely, with the exception of two single-amino acid fragments and one two-amino acid fragment, and the gene sequence reported previously was confirmed. The posttranslational modifications, similar to the ones reported previously for bovine and rat rhodopsin, were also identified. Although porcine rhodopsin has a high degree of homology to bovine and rat rhodopsins and most of their posttranslational modifications are identical, the glycosylation and phosphorylation patterns observed were different. These results show that rhodopsin from a single porcine eye can be characterized completely by MS. This technology opens the possibility of rhodopsin structural and functional studies aided by powerful mass spectrometric analysis, using the fellow eye as an internal control.
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